Project Summary The development of the nervous system requires the proper differentiation, migration, morphogenesis and maturation of neurons. The morphological differentiation of individual neurons and assembly of the trillions of neuronal connections that compose the human nervous system occurs through guided extension of axons and dendrites. The long-term objective of our research is to better understand the intracellular signaling cascades and effector mechanisms that are responsible for axon outgrowth and guidance in the developing brain. For this we must understand how nerve growth cones detect, integrate and respond to soluble, as well as cell- and substratum-associated guidance molecules in their environment. Mutations in genes involved in the detection and transduction of axon guidance information into directed neurite outgrowth are responsible for many neuro- developmental disorders, including autisms, dyslexias, psychological disorders and cognitive deficits. Therefore, our work aimed at better understanding the molecular basis of normal neural development, may help inform treatments for conditions leading to abnormal neural network assembly. While extensive studies have investigated the molecular mechanisms that regulate axon guidance over two- dimensional substrata in vitro or along axonal tracks in vivo, little is known about the signals that control axon guidance across three-dimensional tissues. Our preliminary and recently published data suggest that along with planar filopodia and lamellipodia, growth cones generate orthogonal protrusions in vitro and in vivo that resemble podosomes or invadopodia. Podosomes and invadopodia, collectively referred to as invadosomes, are actin-based cellular protrusions associated with extracellular matrix (ECM) degradation and tissue invasion. We hypothesize that growth cone invadosomes function to actively detect ligands through receptor interactions that regulate actin polymerization and participate in ligand and receptor degradation to modulate ligand-mediated guidance. The three aims of this proposal will use a series of molecular gain of function and loss of function manipulations, together with super resolution three-dimensional fluorescence imaging, to assess the signals that control invadosome formation and their roles in controlling neural network formation.